Liver-Specific, but Not Retina-Specific, Hepcidin Knockout Causes Retinal Iron Accumulation and Degeneration.

The liver secretes hepcidin (Hepc) into the bloodstream to reduce blood iron levels. Hepc accomplishes this by triggering degradation of the only known cellular iron exporter ferroportin in the gut, macrophages, and liver. We previously demonstrated that systemic Hepc knockout (HepcKO) mice, which have high serum iron, develop retinal iron overload and degeneration. However, it was unclear whether this is caused by high blood iron levels or, alternatively, retinal iron influx that would normally be regulated by retina-produced Hepc. To address this question, retinas of liver-specific and retina-specific HepcKO mice were studied. Liver-specific HepcKO mice had elevated blood and retinal pigment epithelium (RPE) iron levels and increased free (labile) iron levels in the retina, despite an intact blood-retinal barrier. This led to RPE hypertrophy associated with lipofuscin-laden lysosome accumulation. Photoreceptors also degenerated focally. In contrast, there was no change in retinal or RPE iron levels or degeneration in the retina-specific HepcKO mice. These data indicate that high blood iron levels can lead to retinal iron accumulation and degeneration. High blood iron levels can occur in patients with hereditary hemochromatosis or result from use of iron supplements or multiple blood transfusions. Our results suggest that high blood iron levels may cause or exacerbate retinal disease.

Ferroportin-mediated iron export from vascular endothelial cells in retina and brain.

Retinal iron accumulation has been implicated in the pathogenesis of age-related macular degeneration (AMD) and other neurodegenerative diseases. The retina and the brain are protected from the systemic circulation by the blood retinal barrier (BRB) and blood brain barrier (BBB), respectively. Iron levels within the retina and brain need to be tightly regulated to prevent oxidative injury. The method of iron entry through the retina and brain vascular endothelial cells (r&bVECs), an essential component of the BRB and BBB, is not fully understood. However, localization of the cellular iron exporter, ferroportin (Fpn), to the abluminal membrane of these cells, leads to the hypothesis that Fpn may play an important role in the import of iron across the BRB and BBB. To test this hypothesis, a mouse model with deletion of Fpn within the VECs in both the retina and the brain was developed through tail vein injection of AAV9-Ple261(CLDN5)-icre to both experimental Fpnf/f, and control Fpn+/+ mice at P21. Mice were aged to 9 mo and changes in retinal and brain iron distribution were observed. In vivo fundus imaging and quantitative serum iron detection were used for model validation. Eyes and brains were collected for immunofluorescence. Deletion of Fpn from the retinal and brain VECs leads to ferritin-L accumulation, an indicator of elevated iron levels, in the retinal and brain VECs. This occurred despite lower serum iron levels in the experimental mice. This result suggests that Fpn normally transfers iron from retinal and brain VECs into the retina and brain. These results help to better define the method of retina and brain iron import and will increase understanding of neurodegenerative diseases involving iron accumulation.

Increased serum proteins in non-exudative AMD retinas.

The blood retinal barrier (BRB) closely regulates the retinal microenvironment. Its compromise leads to the accumulation of retinal fluid containing potentially harmful plasma components. While eyes with non-exudative age-related macular degeneration (AMD) were previously felt to have an intact BRB, we propose that the BRB in non-exudative AMD eyes may be subclinically compromised, allowing entry of retina-toxic plasma proteins. We test this hypothesis by measuring retinal levels of abundant plasma proteins that should not cross the intact BRB. Two cohorts of frozen, post mortem neurosensory retinas were studied by Western analysis. One cohort from Alabama had 4 normal controls and 4 eyes with various forms of AMD. Another cohort from Minnesota had 5 intermediate AMD eyes and 5 normals. Both cohorts were age/post mortem interval (PMI) matched. The non-exudative AMD retinas in the Alabama cohort had significantly higher levels of albumin and complement component 9 (C9) than normal controls. The positive control exudative AMD donor retina had higher levels of all but one serum protein. In both macular and peripheral neurosensory retina samples, intermediate AMD retinas in the Minnesota cohort had significantly higher levels of albumin, fibrinogen, IgG, and C9 than controls. Our results suggest that there may be moderate subclinical BRB leakage in non-exudative AMD. Potentially harmful plasma components including complement or iron could enter the neurosensory retina in AMD patients prior to advanced disease. Thus, therapies aiming to stabilize the BRB might have a role in the management of non-exudative AMD.

Cortical parvalbumin GABAergic deficits with α7 nicotinic acetylcholine receptor deletion: implications for schizophrenia.

Dysfunction of cortical parvalbumin (PV)-containing GABAergic interneurons has been implicated in cognitive deficits of schizophrenia. In humans microdeletion of the CHRNA7 (α7 nicotinic acetylcholine receptor, nAChR) gene is associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia while in mice similar deletion causes analogous abnormalities including impaired attention, working-memory and learning. However, the pathophysiological roles of α7 nAChRs in cortical PV GABAergic development remain largely uncharacterized. In both in vivo and in vitro models, we identify here that deletion of the α7 nAChR gene in mice impairs cortical PV GABAergic development and recapitulates many of the characteristic neurochemical deficits in PV-positive GABAergic interneurons found in schizophrenia. α7 nAChR null mice had decreased cortical levels of GABAergic markers including PV, glutamic acid decarboxylase 65/67 (GAD65/67) and the α1 subunit of GABAA receptors, particularly reductions of PV and GAD67 levels in cortical PV-positive interneurons during late postnatal life and adulthood. Cortical GABAergic synaptic deficits were identified in the prefrontal cortex of α7 nAChR null mice and α7 nAChR null cortical cultures. Similar disruptions in development of PV-positive GABAergic interneurons and perisomatic synapses were found in cortical cultures lacking α7 nAChRs. Moreover, NMDA receptor expression was reduced in GABAergic interneurons, implicating NMDA receptor hypofunction in GABAergic deficits in α7 nAChR null mice. Our findings thus demonstrate impaired cortical PV GABAergic development and multiple characteristic neurochemical deficits reminiscent of schizophrenia in cortical PV-positive interneurons in α7 nAChR gene deletion models. This implicates crucial roles of α7 nAChRs in cortical PV GABAergic development and dysfunction in schizophrenia and other neuropsychiatric disorders.

Cortical synaptic NMDA receptor deficits in α7 nicotinic acetylcholine receptor gene deletion models: implications for neuropsychiatric diseases.

Microdeletion of the human CHRNA7 gene (α7 nicotinic acetylcholine receptor, nAChR) as well as dysfunction in N-methyl-d-aspartate receptors (NMDARs) have been associated with cortical dysfunction in a broad spectrum of neurodevelopmental and neuropsychiatric disorders including schizophrenia. However, the pathophysiological roles of synaptic vs. extrasynaptic NMDARs and their interactions with α7 nAChRs in cortical dysfunction remain largely uncharacterized. Using a combination of in vivo and in vitro models, we demonstrate that α7 nAChR gene deletion leads to specific loss of synaptic NMDARs and their coagonist, d-serine, as well as glutamatergic synaptic deficits in mouse cortex. α7 nAChR null mice had decreased cortical NMDAR expression and glutamatergic synapse formation during postnatal development. Similar reductions in NMDAR expression and glutamatergic synapse formation were revealed in cortical cultures lacking α7 nAChRs. Interestingly, synaptic, but not extrasynaptic, NMDAR currents were specifically diminished in cultured cortical pyramidal neurons as well as in acute prefrontal cortical slices of α7 nAChR null mice. Moreover, d-serine responsive synaptic NMDAR-mediated currents and levels of the d-serine synthetic enzyme serine racemase were both reduced in α7 nAChR null cortical pyramidal neurons. Our findings thus identify specific loss of synaptic NMDARs and their coagonist, d-serine, as well as glutamatergic synaptic deficits in α7 nAChR gene deletion models of cortical dysfunction, thereby implicating α7 nAChR-mediated control of synaptic NMDARs and serine racemase/d-serine pathways in cortical dysfunction underlying many neuropsychiatric and neurodevelopmental disorders, particularly those associated with deletion of human CHRNA7.

Oxidative stress induces lysosomal membrane permeabilization and ceramide accumulation in retinal pigment epithelial cells.

Oxidative stress has been implicated in the pathogenesis of age-related macular degeneration, the leading cause of blindness in older adults, with retinal pigment epithelium (RPE) cells playing a key role. To better understand the cytotoxic mechanisms underlying oxidative stress, we used cell culture and mouse models of iron overload, as iron can catalyze reactive oxygen species formation in the RPE. Iron-loading of cultured induced pluripotent stem cell-derived RPE cells increased lysosomal abundance, impaired proteolysis and reduced the activity of a subset of lysosomal enzymes, including lysosomal acid lipase (LIPA) and acid sphingomyelinase (SMPD1). In a liver-specific Hepc (Hamp) knockout murine model of systemic iron overload, RPE cells accumulated lipid peroxidation adducts and lysosomes, developed progressive hypertrophy and underwent cell death. Proteomic and lipidomic analyses revealed accumulation of lysosomal proteins, ceramide biosynthetic enzymes and ceramides. The proteolytic enzyme cathepsin D (CTSD) had impaired maturation. A large proportion of lysosomes were galectin-3 (Lgals3) positive, suggesting cytotoxic lysosomal membrane permeabilization. Collectively, these results demonstrate that iron overload induces lysosomal accumulation and impairs lysosomal function, likely due to iron-induced lipid peroxides that can inhibit lysosomal enzymes.

Ferrous but not ferric iron sulfate kills photoreceptors and induces photoreceptor-dependent RPE autofluorescence.

Iron has been implicated in the pathogenesis of retinal degenerative diseases, including ocular siderosis. However, the mechanisms of iron-induced retinal toxicity are incompletely understood. Previous work shows that intravitreal injection of Fe2+ leads to photoreceptor (PR) oxidative stress, resulting in PR death within 14 days, and cones are more susceptible than rods to iron-induced oxidative damage. In order to further investigate the mechanism of intravitreal iron-induced retinal toxicity and shed light on mechanisms of iron-induced retinopathy in other mouse models, Fe2+, Fe3+, or saline were injected into the vitreous of adult wild-type mice. Pre-treatment with Ferrostatin-1 was used to investigate whether iron-induced retinal toxicity resulted from ferroptosis. Color and autofluorescence in vivo retinal imaging and optical coherence tomography were performed on day 2 and day 7 post-injection. Eyes were collected for quantitative PCR and Western analysis on day 1 and for immunofluorescence on both day 2 and 7. In vivo imaging and immunofluorescence revealed that Fe2+, but not Fe3+, induced PR oxidative damage and autofluorescence on day 2, resulting in PR death and retinal pigment epithelial cell (RPE) autofluorescence on day 7. Quantitative PCR and Western analysis on day 1 indicated that both Fe2+ and Fe3+ induced iron accumulation in the retina. However, only Fe2+ elevated levels of oxidative stress markers and components of ferroptosis in the retina, and killed PRs. Ferrostatin-1 failed to protect the retina from Fe2+-induced oxidative damage. To investigate the mechanism of Fe2+-induced RPE autofluorescence, rd10 mutant mice aged 6 weeks, with almost total loss of PRs, were given intravitreal Fe2+ or Fe3+ injections: neither induced RPE autofluorescence. This result suggests Fe2+-induced RPE autofluorescence in wild-type mice resulted from phagocytosed, oxidized outer segments. Together these data suggest that intraretinal Fe2+ causes PR oxidative stress, leading to PR death and RPE autofluorescence.

Iron Accumulates in Retinal Vascular Endothelial Cells But Has Minimal Retinal Penetration After IP Iron Dextran Injection in Mice.

Purpose: Iron supplementation therapy is used for iron-deficiency anemia but has been associated with macular degeneration in a 43-year-old patient. Iron entry into the neurosensory retina (NSR) can be toxic. It is important to determine conditions under which serum iron might cross the blood retinal barrier (BRB) into the NSR. Herein, an established mouse model of systemic iron overload using high-dose intraperitoneal iron dextran (IP FeDex) was studied. In addition, because the NSR expresses the iron regulatory hormone hepcidin, which could limit iron influx into the NSR, we gave retina-specific hepcidin knockout (RS-HepcKO) mice IP FeDex to test this possibility. Methods: Wild-type (WT) and RS-HepcKO mice were given IP FeDex. In vivo retina imaging was performed. Blood and tissues were analyzed for iron levels. Quantitative PCR was used to measure levels of mRNAs encoding iron regulatory and photoreceptor-specific genes. Ferritin and albumin were localized in the retina by immunofluorescence. Results: IP FeDex in both WT and RS-HepcKO mice induced high levels of iron in the liver, serum, retinal vascular endothelial cells (rVECs), and RPE, but not the NSR. The BRB remained intact. Retinal degeneration did not occur. Conclusions: Following injection of high-dose IP FeDex, iron accumulated in the BRB, but not the NSR. Thus, the BRB can shield the NSR from iron delivered in this manner. This ability is not dependent on NSR hepcidin production.

Antigenic and mechanistic characterization of anti-AMPA receptor encephalitis.

OBJECTIVE: Anti-AMPAR encephalitis is a recently discovered disorder characterized by the presence of antibodies in serum or cerebrospinal fluid against the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor. Here, we examine the antigenic specificity of anti-AMPAR antibodies, screen for new patients, and evaluate functional effects of antibody treatment of neurons. METHODS: We developed a fusion protein-based western blotting test for anti-AMPAR encephalitis antibodies. Antibody specificity was also evaluated using immunocytochemistry of HEK293 cells expressing deletion mutants of AMPAR subunits. Purified patient IgG or AMPAR antibody-depleted IgG was applied to live neuronal cultures; amplitude and frequency of miniature excitatory postsynaptic currents (mEPSCs) were measured to evaluate functional effects of antibodies. RESULTS: Using both immunocytochemistry and fusion protein western blots, we defined an antigenic region of the receptor in the bottom lobe of the amino terminal domain. Additionally, we used fusion proteins to screen 70 individuals with neurologic symptoms of unknown cause and 44 patients with no neurologic symptoms or symptoms of known neuroimmunological origin for anti-AMPAR antibodies. Fifteen of the 70 individuals had anti-AMPAR antibodies, with broader antigenic reactivity patterns. Using purified IgG from an individual of the original cohort of anti-AMPAR encephalitis patients and a newly discovered patient, we found that application of IgG from either patient cohort caused an AMPAR antibody-dependent decrease in the amplitude and frequency of mEPSCs in cultured neurons. INTERPRETATION: These results indicate that anti-AMPAR antibodies are widespread and functionally relevant; given the robust response of patients to immunomodulation, this represents a significant treatable patient population.